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Evaluating Feruloyl Esterase—Xylanase Synergism For agronomy Article Evaluating Feruloyl Esterase—Xylanase Synergism for Hydroxycinnamic Acid and Xylo-Oligosaccharide Production from Untreated, Hydrothermally Pre-Treated and Dilute-Acid Pre-Treated Corn Cobs Lithalethu Mkabayi 1 , Samkelo Malgas 1 , Brendan S. Wilhelmi 2 and Brett I. Pletschke 1,* 1 Enzyme Science Programme (ESP), Department of Biochemistry and Microbiology, Rhodes University, Grahamstown, Eastern Cape 6140, South Africa; [email protected] (L.M.); [email protected] (S.M.) 2 Department of Biochemistry and Microbiology, Rhodes University, Grahamstown, Eastern Cape 6140, South Africa; [email protected] * Correspondence: [email protected]; Tel.: +27-46-6038081 Received: 4 April 2020; Accepted: 30 April 2020; Published: 13 May 2020 Abstract: Agricultural residues are considered the most promising option as a renewable feedstock for biofuel and high valued-added chemical production due to their availability and low cost. The efficient enzymatic hydrolysis of agricultural residues into value-added products such as sugars and hydroxycinnamic acids is a challenge because of the recalcitrant properties of the native biomass. Development of synergistic enzyme cocktails is required to overcome biomass residue recalcitrance, and achieve high yields of potential value-added products. In this study, the synergistic action of two termite metagenome-derived feruloyl esterases (FAE5 and FAE6), and an endo-xylanase (Xyn11) from Thermomyces lanuginosus, was optimized using 0.5% (w/v) insoluble wheat arabinoxylan (a model substrate) and then applied to 1% (w/v) corn cobs for the efficient production of xylo-oligosaccharides (XOS) and hydroxycinnamic acids. The enzyme combination of 66% Xyn11 and 33% FAE5 or FAE6 (protein loading) produced the highest amounts of XOS, ferulic acid, and p-coumaric acid from untreated, hydrothermal, and acid pre-treated corn cobs. The combination of 66% Xyn11 and 33% FAE6 displayed an improvement in reducing sugars of approximately 1.9-fold and 3.4-fold for hydrothermal and acid pre-treated corn cobs (compared to Xyn11 alone), respectively. The hydrolysis product profiles revealed that xylobiose was the dominant XOS produced from untreated and pre-treated corn cobs. These results demonstrated that the efficient production of hydroxycinnamic acids and XOS from agricultural residues for industrial applications can be achieved through the synergistic action of FAE5 or FAE6 and Xyn11. Keywords: Ferulic acid; Feruloyl esterase; Xylanase; Synergy; Xylo-oligosaccharides 1. Introduction Lignocellulosic biomass is widely considered one of the most promising low-cost feedstocks for the production of renewable energy and value-added chemicals. It is primarily composed of three major components, namely: cellulose (40–60%), hemicellulose (20–40%), and lignin (10–25%) [1]. Waste agricultural residues generated during crop harvesting and processing (e.g., rice straw, wheat straw, corn stover, corn cobs, sugarcane bagasse, sorghum bagasse, etc.) are renewable biomass resources that are readily available and inexpensive [2]. A significant amount of research has focused on developing environmentally friendly methods of utilising lignocellulosic biomass. In this regard, enzymatic conversion has emerged as a major technological platform that offers several advantages Agronomy 2020, 10, 688; doi:10.3390/agronomy10050688 www.mdpi.com/journal/agronomy Agronomy 2020, 10, 688 2 of 14 such as environmental benefits and lower energy costs, with no formation of undesirable by-products. Due to the complexity and heterogeneity in the structure of biomass, its enzymatic conversion requires the synergistic cooperation of several enzymes [3]. A detailed understanding of this synergistic cooperation is key in the development of enzyme cocktails for the optimal production of value-added chemicals from lignocellulosic biomass. Feruloyl esterases (FAEs, EC 3.1.1.73) and endo-xylanases (EC 3.2.1.8) are essential enzymes for the degradation of xylan, the most abundant hemicellulose in biomass. FAEs catalyze the cleavage of covalent ester linkages between hydroxycinnamic acids and polysaccharides, releasing ferulic acid (FA) and p-coumaric acid (p-CA) from lignocellulosic biomass [4,5]. FA, the most abundant hydroxycinnamic acid, is usually esterified at the C-5 hydroxy group of the arabinofuranosyl units of arabinoxylan of commelinid plants [6]. Hydroxycinnamic acids are used in the food industry as precursors for vanillin, p-hydroxybenzoic acid and 4-vinylphenol production, and as food preservatives because of their antimicrobial properties [7], while in the pharmaceutical industry, they can be used for their antioxidant and anti-inflammatory properties [8–11]. Application of FAEs not only releases hydroxycinnamic acid, but also decreases biomass recalcitrance, making it more accessible for further hydrolysis by carbohydrate-active enzymes [11]. Endo-xylanases are essential in degrading xylan as they catalyze the random cleavage of β-1,4-D-xylosidic linkages, generating xylo-oligosaccharides (XOS) [12]. The enzymes have been classified into glycoside hydrolase (GH) families 5, 8, 10, 11, 30, 43, 62 and 98, with GH10 and 11 being the two families that have been extensively characterized (www.cazy.org). Endo-xylanase generated XOS from agricultural residues are used in food, feed, and pharmaceutical industries due to their prebiotic and antioxidant activity [13,14]. FAEs exhibit a synergistic interaction with xylanases during the hydrolysis of a range of lignocellulosic substrates, which is demonstrated by improved yields in the production of XOS and FA. Xylanases generate ferulated XOS, which become preferred substrates for FAEs to cleave ester bonds from, liberating FA as a product [15]. The removal of FA then increases the accessibility of xylanases to XOS for further hydrolysis into shorter XOS and/or xylose. Studies have reported significant increases in the amount of FA released from arabinoxylan after the enzymatic hydrolysis by FAEs in the presence of xylanases [16–18]. Although FAEs from different microorganisms have been used for the co-production of FA and XOS, some of these FAEs show limited hydrolysis efficiencies. Therefore, novel FAEs with exceptional catalytic properties are still required for the formulation of more efficient enzyme cocktails. Rashamuse and co-workers [19] functionally screened novel FAEs from the hindgut prokaryotic symbionts of Trinervitermes trinervoides termite species, but the application of these enzymes in degrading agricultural residues has not yet been explored. In this study, the synergistic action of two new termite metagenome derived FAEs (FAE5 and FAE6) and a GH11 xylanase from Thermomyces lanuginosus was optimized on insoluble wheat arabinoxylan (model substrate) and then applied to corn cobs (a natural substrate) for the production of XOS and hydroxycinnamic acids. The study presented demonstrated that high quantities of XOS, p-CA and FA were generated from corn cobs as a result of synergistic interactions between Xyn11 and FAE5 or FAE6. 2. Materials and Methods 2.1. Chemicals, Substrates and Enzymes Escherichia coli BL21 (DE3) was used for the expression of fae5 and fae6 genes harboured in pET28 plasmids. The cells were cultured in Luria-Bertani (LB) medium containing 50 µg/mL kanamycin at 37 ◦C. The induction was conducted by following the method described previously [20]. Enzymes were purified using 5 mL Ni-NTA Superflow Cartridges (QIAGEN®), purchased from QIAGEN (Germany), as per the manufacturer’s instructions. Xylanase (Xyn11) from Thermomyces lanuginosus and ethyl ferulate (EFA) were purchased from Sigma Aldrich (South Africa). Insoluble wheat arabinoxylan (WAX) was purchased from Megazyme™ (Ireland). Agronomy 2020, 10, 688 3 of 14 2.2. Pre-Treatment of Corn Cobs Milled corn cobs (CC) was treated using hydrothermal pre-treatment or dilute acid pre-treatment. A total of 10 g of biomass was suspended in Milli-Q water (for hydrothermal treatment) or in a 0.5% (w/w) sulphuric acid solution (for dilute acid treatment) (solid: liquid ratio of 1:10) and autoclaved for 20 min at 121 ◦C. The pre-treated CC slurry was filtered, followed by washing the solids repeatedly with Milli-Q water and then oven drying to constant weight at 50 ◦C for 48 h. 2.3. Chemical Characterization of CC The total carbohydrates of CC were determined in triplicate by using a modified sulphuric acid method described previously [21]. Briefly, 300 mg of CC (untreated and pre-treated) was hydrolyzed with 72% (v/v) sulphuric acid at 30 ◦C for 1 h, diluted to 3% (v/v) sulphuric acid and autoclaved to solubilize the carbohydrate fraction. Following the hydrolysis, fractions were filtered to remove the insoluble lignin from the solution. Determination of the alkali-extractable hydroxycinnamic acid content was carried out by treating 10 mg of biomass with 1 M NaOH solution for 24 h at room temperature and in the dark. The liquors obtained from alkaline treatments were separated from the solid fraction by centrifugation at 16,000 g × for 5 min. The liquors were neutralized by 2 volumes of 1 M HCl and analyzed by HPLC as described in Section 2.4. The morphological structure of untreated and pre-treated biomass was analyzed with a scanning electron microscope (SEM), JOEL JSM 840. CC samples were mounted on a metal stub with adhesive tape and coated with a thin layer of gold prior to SEM analysis. In order to detect changes in functional groups, FTIR analysis of untreated and pre-treated CC was conducted by loading a few milligrams
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